The detection of immobilized nucleic acid, especially nucleic acid separated on gels, is a widely used method. Numerous chromophores and dyes exist for the detection of nucleic acids however, despite its relatively high detection limit, ethidium bromide is still one of the most commonly used nucleic acid detection reagents due in part to its ease of use and low cost.
Ethidium bromide is easy to use as a nucleic acid gel stain because the nucleic acid can be pre- or post-stained and requires no special equipment for visualization beyond a UV light source. Ethidium bromide is excited by UV light, less than 400 nm, and has an emission spectra of about 620 nm when bound to DNA. Thus, the stained gels can be excited by an ultraviolet transilluminator, which typically has a light wavelength of about 300 nm, and the excited ethidium bromide-DNA complex gel photographed using black and white Polaroid film. Despite the convenience of ethidium bromide, the compound posses some significant disadvantages; namely that the compound is a known mutagen and carcinogen which requires special handling and waste disposal procedures. Ethidium bromide has been shown to inhibit replication in several organisms by interfering with both DNA and RNA synthesis, to be mutagenic in an Ames test and to cause frameshift mutations in bacteria (M. J. Waring J. Mol. Biol. 13 (1965) 269-282; McCann et al. PNAS 72 (1975) 5135-5139; Singer et al. Mutation Research 439 (1999) 37-47). This is because ethidium bromide is believed to intercalate dsDNA and thus causes errors during replication (Fukunaga et al. Mutation Research 127 (1984) 31-37).
Due to these limitations of ethidium bromide, we wanted to develop an improved method for detecting immobilized nucleic acid that retained the advantages of ethidium bromide, ease of use and low cost, but overcame the limitations of ethidium bromide. Thus, to satisfy this criteria the method and subsequent dye must 1) be relatively easy to synthesize in large quantities (low cost), 2) be present in the staining solution at a relatively low concentration (low cost), 3) excited by UV light (ease of use so that the nucleic acid-dye complex can be visualized with a UV transilluminator), 4) at least as sensitive as ethidium bromide (ease of use), 5) non-genotoxic (non-mutagenic and non-toxic) and 6) non-hazardous to aquatic life thus requiring no special waste disposal.
Here in we report the use of a class of unsymmetrical cyanine dye compounds (U.S. Pat. Nos. 4,883,867 and 4,957,870) for detecting immobilized nucleic acid polymers that is at least as sensitive as ethidium bromide, requires no additional reagents or instruments than ethidium bromide and can be made in large quantities. We also report on a compound in this class of dye compounds that is non-genotoxic and therefore requires no special handling or waste disposal procedures by the end user. Thus, this present invention is an improvement over currently used nucleic acid detection reagents and solves a problem not previously solved.